Distintos Blancos Contra Ticks

Parasite Immunology, 2016, 0, 1–16 DOI: 10.1111/pim.
12339
Commissioned Review Article
Strategies for new and improved vaccines against ticks and

tick-borne diseases
CEK,
J. DE LA FUENTE,1,2,† P. KOPA 3,†
A. LEW-TABOR4,5,† & C. MARITZ-OLIVIER6,†
1
on en Recursos Cinegeticos IREC (CSIC-UCLM-JCCM), Ciudad Real, Spain, 2Department of Veterinary
SaBio, Instituto de Investigaci
Pathobiology, Center for Veterinary Health Sciences, Oklahoma State University, Stillwater, OK, USA, 3Institute of Parasitology, Biology
Centre Czech Academy of Sciences, Ceske Budejovice, Czech Republic, 4Queensland Alliance for Agriculture & Food Innovation, The
University of Queensland, St. Lucia, Qld, Australia, 5Centre for Comparative Genomics, Murdoch University, Perth, WA, Australia,
6
Department of Genetics, Faculty of Natural and Agricultural Sciences, University of Pretoria, Pretoria, South Africa
SUMMARY Keywords interactomics, reverse genetics, systems biology,

tick, vaccine, vaccinology
Ticks infest a variety of animal species and transmit pathogens
causing disease in both humans and animals worldwide.
Tick–host–pathogen interactions have evolved through
INTRODUCTION
dynamic processes that accommodated the genetic traits of
the hosts, pathogens transmitted and the vector tick species Diseases caused by arthropod-borne pathogens account
that mediate their development and survival. New for over 20% of all emerging infectious diseases recorded
approaches for tick control are dependent on defining molec- between 1940 and 2004 (1). Ticks are considered to be sec-
ular interactions between hosts, ticks and pathogens to allow ond worldwide to mosquitoes as vectors of human diseases
for discovery of key molecules that could be tested in vacci- and the most important vectors of diseases that affect ani-
nes or new generation therapeutics for intervention of mal health worldwide, with the largest effect on the cattle
tick–pathogen cycles. Currently, tick vaccines constitute an industry (2, 3). It is therefore evident that ticks and their
effective and environmentally sound approach for the control associated transmitted pathogens constitute a growing bur-
of ticks and the transmission of the associated tick-borne den for human and animal health worldwide (4, 5). Tick
diseases. New candidate protective antigens will most likely control has been primarily based on the use of chemical
be identified by focusing on proteins with relevant biological acaricides resulting in partial efficacy and enhanced selec-
function in the feeding, reproduction, development, immune tion of resistant tick populations (6). Therefore, new
response, subversion of host immunity of the tick vector and/ approaches are needed for effective control of ticks and
or molecules vital for pathogen infection and transmission. their associated tick-borne diseases (TBDs), of which vac-
This review addresses different approaches and strategies cines appear to be an effective and environmentally sound
used for the discovery of protective antigens, including focus- option (6).
ing on relevant tick biological functions and proteins, reverse The only commercially available vaccines against
genetics, vaccinomics and tick protein evolution and interac- ectoparasites were developed and registered in the early
tomics. New and improved tick vaccines will most likely con- 1990s for the control of cattle tick infestations (7). These
tain multiple antigens to control tick infestations and vaccines are based on recombinant Rhipicephalus micro-
pathogen infection and transmission. plus BM86/BM95 antigens and demonstrated the impor-
tant advantages of being a cost-effective and
Correspondence: Jose de la Fuente, SaBio, Instituto de Investi- environmentally friendly alternative with a dual effect of
gacion en Recursos Cinegeticos IREC-CSIC-UCLM-JCCM, reducing tick infestations and preventing ticks from trans-
Ronda de Toledo s/n, 13005 Ciudad Real, Spain mitting disease-causing pathogens (7). The protective
(e-mail: jose_delafuente@yahoo.com). mechanism characterized for BM86/BM95 tick vaccines is
†
All authors contributed equally to this article.
based on the development of antigen-specific antibodies in
Disclosures: None.
Received: 30 January 2016 immunized hosts that interact and subsequently affect the
Accepted for publication: 13 May 2016 biological function of the targeted antigen within the tick
© 2016 John Wiley & Sons Ltd 1

J. de la Fuente et al. Parasite Immunology
tissues, impairing tick feeding on the immunized hosts targets (Achilles’ heels) that could be affected by anti-
(7–9). The application of the BM86/BM95-based vaccines bodies present in vaccinated hosts. In this section, we will
results in reduction of the number, weight and repro- enumerate the physiological processes that are targeted to
ductive capacity of engorging female ticks, resulting in date (Figure 1) and link the research findings to our cur-
a gradual reduction in tick infestations in subsequent rent understanding and approaches for the development of
generations (7). efficacious tick vaccines.
The application of commercial vaccines for the control
of cattle ticks demonstrated that tick vaccines also consti-
Tick attachment and on-host feeding
tute an effective and environmentally sound approach for
the control of TBDs (7). However, despite recent advances Blocking attachment of a tick to the host would be the
in the identification and characterization of candidate tick ultimate goal, impairing not only tick feeding but also
vaccine antigens, new tick and pathogen-derived protective transmission of tick-borne pathogens. The process of
antigens need to be identified to increase vaccine efficacy attachment starts with the intrusion of the tick mouthpart
for the control of TBDs (6, 10). Herein, we review some (hypostome) into the host dermis, followed by anchoring
of the major approaches and strategies that are currently of the tick by a cement cone that is mostly comprises of
being used for the identification of protective antigens, as secreted glycine-rich proteins (11).
well as some future trends to develop improved tick vac- Several cement cone antigens have been identified based
cines. on their immunogenicity. Screening of a cDNA expression
library from Haemaphysalis longicornis females, with rab-
bit immune serum raised against tick saliva, resulted in
FROM TICK BIOLOGY TO TICK VACCINES: A
the identification of a collagen-like protein P29 (12) and
RATIONAL APPROACH
an unannotated protein HL 34 (that is rich in tyrosine and
The rational approach towards discovery of promising proline residue repeats) (13). Immunization of rabbits with
vaccine candidates that will impair biological processes recombinant P29 caused about 50% mortality of H. longi-
stems from our knowledge of specific features in tick phys- cornis larvae and nymphs (12). Immunization with recHL
iology that evolved during the adaptation of an ancestor 34 reduced survival of both immature and adult H. longi-
mite to an obligatory blood-feeding lifestyle. Therefore, cornis stages. The appearance of ticks fed on vaccinated
the processes were associated with tick attachment to the hosts points to impaired blood meal uptake and digestion
host, followed by undisturbed feeding on the host, intra- (13).
cellular digestion of the large amount of ingested blood Two vaccine candidates were identified from the cement
and finally, processing of the blood meal into huge cone of the African brown ear tick, Rhipicephalus appen-
clutches of eggs laid by the engorged females. To date, this diculatus. A highly immunogenic glycine-rich protein RIM
approach has offered a number of potential vulnerable 36 has the potential to be developed into a diagnostic
Figure 1 An overview of tick physiological

processes and involved molecules tested as
vaccine candidates. For abbreviations, tick
species and references, see the corresponding
paragraphs in the text. Underlined – the
most promising rationally designed vaccine
candidates.
2 © 2016 John Wiley & Sons Ltd, Parasite Immunology, 0, 1–16

Volume **, Number **, ** ** Strategies for new and improved vaccines
marker for detection of cattle that have been exposed to Metis 1. Vaccination had no effect on tick attachment and
feeding ticks; however, its protective effect as a vaccine has mortality, but reduced the engorgement weight of I. ricinus
not been assessed (14). In contrast, a secreted 15 kDa pro- females and their ability to lay eggs (20). A slight increase
tein called 64P seems to be invisible to the host immune in mortality (some 15%) was reported for H. longicornis
response most likely given its similarity to the mammalian nymphs and adults fed on rabbits vaccinated with recom-
skin keratins (15). The N-terminal portion of 64P displays binant metalloprotease HLMP1 (21). Most recently, pro-
similarity with the a2 chain of collagen type IV and exhib- tective potential of the R. microplus metalloprotease
ited very promising protection in animal trials when BrRm-MP4 against R. microplus was tested in cattle trials.
expressed as truncated 64P constructs (64TRPs) with glu- The overall efficacy of vaccination with recombinant
tathione S-transferase (GST) as a fusion protein. The vac- BrRm-MP4 was 60%, mainly reducing the number of
cine based on 64TRPs was reported to have a ‘dual engorged ticks that completed feeding on immunized cattle
action’ as tick feeding on immunized animals was primar- (22).
ily impaired at the skin feeding site, but cross-reaction of
anti-64TRP antibodies with midgut antigenic epitopes also
Protease inhibitors
caused rupture of the tick midgut and death of engorged
ticks (15). In addition, several 64TRPs exhibited a cross- A number of host defence reactions such as blood coagu-
protective response against other tick species, namely lation and complement activation are mediated by cas-
Rhipicephalus sanguineus and Ixodes ricinus, underscoring cades of serine proteases (23). Recombinant serine
their potential as a broad-spectrum tick vaccine (16). protease inhibitors (serpins) from several tick species have
Intriguingly, it was later demonstrated in a mouse model been examined as candidate vaccine targets. HLS2 from
that vaccination with 64TRPs antigens protected mice H. longicornis resulted in some 40% mortality of nymphs
against lethal transmission of tick-borne encephalitis virus and adults fed on immunized rabbits (24, 25). Four ser-
by still unknown mechanisms (17). However, these results pins, termed RAS-1-4 identified from R. appendiculatus
were obtained in rodent models (guinea pigs and mice), (26), were tested either as a mix of recombinant RAS-1
with no evidence that it is effective in protection against and RAS-2 (27) or as a cocktail composed of three anti-
tick infestation in natural hosts such as cattle. gens, RAS-3, RAS-4 and the immune-dominant RIM36
(28). Immunization of cattle with recombinant RAS-1 and
RAS-2 reduced the number of engorged R. appendiculatus
Proteases and protease inhibitors at the tick–host
nymphs by 60% and increased mortality of adult males
interface
and females by 43% and 28%, respectively (27). A combi-
Tick saliva is an extremely rich cocktail of bioactive com- nation of RAS-3, RAS-4 and RIM6 mainly affected the
pounds that facilitates ixodid ticks to feed undisturbed on mortality of Theileria parva-infected females (~50%) (28).
the host for several days. Our knowledge about the mole- The recombinant serpin, IRIS from I. ricinus, exerted a
cules produced by tick salivary glands (sialomes) increased significant protective immunity against nymphs and adults
dramatically during the past decade due to high-through- fed on vaccinated rabbits as displayed by increases in mor-
put transcriptome-based approaches (for review, see Ref. tality (~30%), reduced weight of engorged ticks and pro-
(18)). Most of the bioactive compounds modulating host longed feeding time. No effect was obtained for nymphs
haemostasis, immune and inflammatory response are fed on vaccinated mice (29).
members of multigene families. This observation may Another group of tick protease inhibitors that are con-
lower the potential of individual saliva molecules as vac- sidered as possible candidate antigens are cystatins that
cine candidates given their extreme redundancy, but other inhibit a variety of cysteine peptidases involved in modula-
possibilities to target these antigens during vaccination are tion of host immunity and inflammation (30). Sera from
discussed later in this article. Despite the great redundancy guinea pigs that were repeatedly exposed to tick infesta-
of these proteins, some promising results have been tion did not recognize sialostatin L2, a secreted
reported for tick proteases and protease inhibitors immunomodulatory cystatin from Ixodes scapularis sali-
involved in tick–host interactions. vary glands. This antigen was subsequently termed as ‘si-
lent’ antigen by the authors (31). It does however need to
be confirmed that this observation is not due to the anti-
Metalloproteases
gen not being processed in antigen presenting cells, vs.
The protective ability of Metis 1, a member of a putative being secreted into an environment where immune-sup-
metalloprotease family from salivary glands of I. ricinus pression is occurring due to tick saliva proteins. After all,
(19), was tested on rabbits immunized with recombinant a vast number of salivary gland proteins are not
© 2016 John Wiley & Sons Ltd, Parasite Immunology, 0, 1–16 3

recognized by immune sera, but they remain immunogenic osmoregulatory system. No vaccination effect with recombi-
upon vaccination. Nevertheless, feeding of I. scapularis nant RmAQP2 has been reported to date.
nymphs on guinea pigs vaccinated with Sialostatin L2 was
impaired as early rejection of the nymphs occurred as well
Blood digestion
as a reduction in the ability of remaining nymphs to
imbibe blood (31). Similar results were reported for the Blood meal processing occurs intracellularly within the
cystatin OmC2 from the soft tick, Ornithodoros moubata. digestive cells of the tick gut epithelium (11) and is per-
Vaccination of mice with recombinant OmC2 significantly formed by an orchestrated network of acidic peptidases
reduced the number of O. moubata first nymphal stage comprising aspartic endopeptidases of the cathepsin D-
capable to complete feeding, resulting in increased post- type, cysteine endo- and exopeptidases of the papain type
engorgement mortality (32). As OmC2 is expressed in (cathepsin L, B and C) and asparaginyl endopeptidases of
both the salivary gland and gut tissues of O. moubata, the the legumaintype (for review, see Ref. (42)). This multi-
authors speculated about a possible ‘dual’ action that enzyme digestive apparatus may seem to offer multiple
impairs both feeding (via interfering with modulation of potential antigens for efficient tick control, as suggested
host immune response) and blood digestion (32). More by the results of RNAi-mediated silencing of individual
recently, identification and characterization of cystatins peptidases in several tick species (42–44). However, to our
from other tick species, namely R. appendiculatus, R. mi- knowledge, no successful vaccination experiment using a
croplus and Ixodes ovatus, were reported and the immuno- recombinant tick digestive enzyme has been reported to
genicity of corresponding recombinants tested as a first this end. Experimental vaccination of rabbits with a cock-
step towards their assessment as tick vaccine candidates tail of recombinant cathepsins D, B, L, C and legumain
(33–35). from I. ricinus has been performed (45). Despite high anti-
body titres against all of the recombinant antigens, only a
slight increase in the mortality of I. ricinus females was
Water balance
observed in two of the three vaccinated rabbits. Currently,
Osmoregulation and water balance play an extremely the potential of targeting multiple tick digestive peptidases
important role in tick physiology as a fully engorged ixodid by vaccination is limited given the high redundancy of the
female tick can imbibe 200–300 times her own weight of system, as previously demonstrated by specific inhibition
host blood. The blood meal is concentrated in the gut with of a subset of enzymes (46). The possibility of targeting
about 70% of the fluid being returned to the host via saliva- unique protein–protein interaction (PPI) sites that may be
tion (11, 36). Therefore, water channels (aquaporins) pre- more evolutionary conserved is discussed later in this
sent another rational target for vaccine development (37, article.
38). The function of aquaporin was examined in I. ricinus
by RNAi silencing of an aquaporin termed IrAQP1, result-
Heme and iron metabolism
ing in 50% weight reduction of semi-engorged females that
fed for 5 days. Vaccination experiments remain to be per- Ticks have to cope with an excess of toxic heme and iron-
formed for IrAQP1 (39). Three aquaporins have been identi- derived metabolites from their blood meal. The majority
fied in the cattle tick R. microplus, and the vaccination of heme is detoxified via its accumulation in organelles
potential of RmAQP1 was recently tested. Two independent called haemosomes (47) of which only a small portion is
pen trials (conducted in Brazil using Holstein-Friesian used for tick’s own metabolic demands as they are not
calves vaccinated with Pichia-expressed recombinant capable of heme biosynthesis (48, 49). The nonheme iron
RmAQP1) exerted a high efficacy with 75% and 68% is sequestered intracellularly by ferritin 1 (Fer1), which is
decrease, respectively, in the number of adult ticks that com- related to mammalian heavy-chain ferritins (50). Their
pleted feeding (40). This result promotes RmAQP1 among expression was shown to be post-transcriptionally regu-
the most promising vaccine candidates that should be fur- lated by an iron-responsive protein (IRP) (51). Transport
ther tested against cattle ticks in different geographic of nonheme iron from the tick gut to peripheral tissues is
regions. More recently, stage and tissue expression profile mediated by a secreted ferritin 2 protein (Fer2) that is dis-
and functional assessment by RNAi were reported for tinct from tick ferritin 1 and mammalian ferritins (51).
R. microplus aquaporin 2 (RmAQP2) (41). RmAQP2 was The critical functions of tick ferritins in tick development
found predominantly expressed in the salivary glands of and reproduction were demonstrated by RNAi-mediated
semi-engorged females, and RNAi-mediated silencing silencing in I. ricinus (51) and H. longicornis (52, 53). To
resulted in a minor increase of the body weight of engorged date, much emphasis has been placed on tick Fer2 as it
females, pointing towards impairment of the displays other desired attributes expected from an ideal

concealed antigen, including: (i) not being present in the mating status. An engorgement factor that is passed from
mammalian host; (ii) it is encoded by a single gene (no males to females during copulation was identified in Ambly-
redundancy problem); (iii) it is expressed in the gut and oma hebraeum and termed voraxin (60). It is composed of
therefore can come into contact with antibodies taken up two unrelated peptides tagged AHEFa and AHEFb of 161
by a blood meal from a vaccinated host; and (iv) it was and 116 kDa, respectively. During vaccination trials in rab-
shown to be highly immunogenic as a recombinant pro- bits using a mixture of recombinant AHEFa and AHEFb,
tein. In the first pilot experiments, vaccination of rabbits only 25% of normally mated A. hebraeum females were cap-
with recombinant Fer2 from I. ricinus substantially able to fully engorge (60). The potential of voraxin as a
reduced tick infestation, weight of engorged females and vaccine candidate was also evaluated against R. appendicu-
their fertility with an overall efficacy of 98% (54). In the latus. Immunization of rabbits with recombinant voraxin a
same study, vaccination of cattle with recombinant Fer2 reduced the weight of engorged R. appendiculatus ticks by
from R. microplus was reported to exert comparable effi- some 40% and also significantly impaired subsequent
cacy of about 60% (which was similar to that of the Bm86 oviposition and larval hatching efficacy (61).
antigen used as positive control) in protection against
R. microplus. Some 70% protection was observed against
Vitellogenesis and fertility
Rhipicephalus annulatus (54). The vaccination potential of
Fer1 and Fer2 from H. longicornis has been evaluated Impairment of vitellogenesis, embryogenesis and fertility
using a rabbit model. Immunization with recombinant by vaccination would be meaningful especially to control
H. longicornis Fer2 resulted in a significant reduction of the population of one-host ticks such as R. microplus.
body weight with reduced oviposition and hatching Three enzymes involved in vitellogenin processing in
observed for both groups immunized with Fer1 and Fer2 R. microplus eggs have been identified and characterized
(55). Most recently, the vaccination potential of other to date. These include two aspartic peptidases, the Boophi-
molecules possibly playing a role in tick heme and iron lus yolk pro-cathepsin (BYC) (62) and tick heme-binding
metabolic pathways of I. ricinus was screened using RNAi aspartic peptidase (63), as well as a cathepsin L-like vitel-
and experimental vaccination of rabbits (56). In addition logenin degrading cysteine endopeptidase (VTDCE) (64).
to Fer1, Fer2 and IRP (see above), RNAi screening also Bovine immunization was performed with native or recom-
evaluated transferrin 2 (Trf2), a divalent metal transporter binant BYC and VTDCE and the individual enzymes con-
1 (Dmt1), ferrochelatase (Fech), heme-binding lipoprotein ferred only limited protection (some 25%) in overall
(HeLp), heme responsive gene 1 (HRG1) and vitellogenins efficacy (65–68). A combination of recombinant BYC and
1 and 2 (Vg1, Vg2). Results from both RNAi and vaccina- VTDCE and the previously mentioned GST from
tion studies concluded that none of these molecules sur- H. longicornis was evaluated as a cocktail vaccine in field
passed Fer2 with regard to their impact on tick conditions (69). The number of semi-engorged females fed
development and reproduction efficiency. on the vaccinated cattle was reduced by about 50%, which
seems to correlate with increased body weight gain in the
vaccinated calves. This desired result further underscores
Detoxification
the potential of multi-antigenic vaccines that might be
Glutathione S-transferases from numerous parasites have capable to provide protection against ticks and maybe also
been shown to play an important role in metabolic detoxi- against the diseases they transmit.
fication of xenobiotics, reactive oxygen species, heme and
other toxic compounds, and as such present as a promis-
REVERSE GENETICS APPROACH TO TICK
ing antigens for antiparasitic vaccines (57, 58). The protec-
VACCINES
tive potential of one such GST was examined using
H. longicornis GST in cattle trials against R. microplus
Tick genomic and sequence resources
infestations (59). Given its cross-reactivity with other tick
proteins, three of four calves immunized with recombinant Innovations in genome sequencing technologies have pro-
H. longicornis GST displayed some 50% reduction of gressed the fields of reverse genetics and reverse vaccinol-
engorged R. microplus adults. ogy (RV). Fewer genome projects have been developed for
tick species due to their large genome sizes and the pres-
ence of dense repetitive regions. For example, the genome
Mating
sizes for I. scapularis and R. microplus were estimated at
The ability of ixodid females to initiate and complete the 21 and 71 Gb, respectively, with repetitive sequences
rapid engorgement phase of feeding is determined by their occurring in a mixture of long and short period

Table 1 Examples of EST and transcriptome sequencing studies for selected tick species
Species Life stages sequenced Technology Output References
Amblyomma Larvae, nymph, adult male, EST Sanger 14 310 ESTs (81)
americanum adult female, engorged female sequencing
Amblyomma Female feeding salivary glands; 454 Roche 11 240; 4604; 3796 (168)
triste, A. nymphs (A. triste only) (coding sequences, CDSs)
parvum,
A. cajennense
Dermacentor Salivary glands from: unfed, 454 Roche 21 769 CDSs (169)
andersoni 2 and 5 day fed
(female adults)
Haemaphysalis Female salivary glands Illumina 54 357 transcripts (170)
flava
Ixodes ricinus Female mid-gut Ion torrent 60 693 transcripts (76)
Haemocytes (females), 454 and Illumina 2860 CDSs (454); (171, 172)
mid-gut, salivary glands 24 687 CDSs (Illumina)
(nymphs, females and males)
Larvae and female salivary Illumina ~over 27 000 transcripts (173)
glands
Ixodes Nymphs and female mid-guts Illumina 17 503 transcripts (114)
scapularis and salivary glands
ISE6 cells Illumina 37 990 transcripts (115)
Rhipicephalus Unfed and fed female Illumina/Solexa 25 113 transcripts; cysteine (174)
haemaphysaloides salivary glands proteases up-regulated in feeding ticks
Rhipicephalus Larvae, nymph, feeding female EST sanger CattleTickBase – 18 Gb of gene (74)
(Boophilus) ticks, female organs, male ticks sequencing; enriched genome regions; assembled
microplus Cot enriched transcriptome (RmiTr Version 10) with
sequences; 454 28 893 sequences (24 673 517 bp)
interspersions with 65–80% of the DNA following a pat- particularly salivary glands (sialome) and/or the mid-guts
tern of short period interspersions (70). Since this publica- (mialome). Although EST libraries have been used to
tion in 2005, the I. scapularis genome assembly was develop custom microarrays to examine up- and down-
reported at 389 coverage from 369 495 scaffolds built regulation of tick genes in different samples (77, 78), this
using 570 637 contigs with a total number of 20 486 cod- review will focus on sequencing technologies only. Table 1
ing genes (GenBank Accession: ABJB000000000.1 Ixodes provides examples of EST and transcriptome sequencing
scapularis strain Wikel colony (71)). A preliminary 18 Gb studies for selected tick species.
of the R. microplus gene enriched genome sequence has
been reported (72). McCooke et al. (73) reported that Cat-
Introduction to reverse genetics and applications to tick
tleTickBase is a database that contains all of the sequences
research
from the R. microplus genome sequencing project, pre-
sently at ~49 coverage with Pac Bio (74). Thus, the Forward genetic methods in eukaryotes have been used for
sequencing of the whole R. microplus genome is proceed- more than 100 years to identify the genes responsible for
ing with only preliminary data published to date including a particular phenotype, reviewed by Hardy et al. (79).
several BAC clones and the mitochondrial genome of the Reverse genetics consists of modifying the activity of a tar-
Deutsch Texas R. microplus strain (73, 75). Recently, pre- get gene to determine the phenotypic consequences. Modi-
liminary genome sequence data for Ixodes ricinus were fications to the genome are implemented by random
reported with ~998 million paired-end reads (~479 cover- chemical or insertional mutagenesis (e.g. transposons or
age, approximately 63% of the total coding sequences) retroviruses), or specifically using homologous recombina-
from Ilumina Inc, San Diego, CA, USA HiSeqTM next- tion of a target gene (79). Other approaches targeting
generation sequencing (GenBank Accession Numbers: mRNA include morpholino antisense oligonucleotides and
JXMZ01000000 and SRP051465) (76). Due to the com- RNA interference (RNAi). RNAi has shown to be effi-
plexity of tick genomes, EST and next-generation tran- cient in post-transcriptional gene silencing using exoge-
scriptome sequencing projects have been undertaken to nous or endogenous double-stranded RNAs to
inform researchers in terms of the identity of specific knockdown specific transcripts (80). For reverse genetics
genes up-regulated by different organs in feeding ticks, approaches, ideally the whole-genome sequence is required

to screen and predict the specificity of these knockdown any time followed by the screening for protective immunity
treatments. In addition, genome sequencing has identified as the ‘rate-limiting’ step (87). Reverse vaccinology has so
genes with unassigned functions. Assigning functions to far been successful for the development of novel antibacte-
these genes can be achieved by predicting subcellular rial vaccines with relatively small genomes in comparison
localizations of gene products and by describing the phe- to ectoparasites such as ticks (88). With the completion of
notypes associated with their disruption (79). Thus, essen- the I. scapularis genome, this provides an opportunity to
tially high-throughput reverse genetic approaches are develop a tick specific RV approach. Approaches devel-
major tools in the post-genomic era. oped to predict vaccine antigens in ticks to date have not
A challenge with tick sequence analysis is the presence of been undertaken using complete whole-genome data. One
a large number of genes whose functions are both unknown study has used a B-cell epitope prediction programme
and unpredicted. These have been called ‘orphan genes’ (BCPREDS) to examine I. scapularis salivary proteins;
which lack detectable sequence homology to genes in pre- however, in vivo confirmation of the immunogenicity of
existing databases (81). For ticks, the most exploited reverse these proteins was not demonstrated (89). In addition, 24-
genetic approach has been RNAi (82). For the most part, h fed I. scapularis tick saliva proteins were expressed using
tick researchers have developed dsRNA treatments in the phage display and panned using antibodies to identify
absence of complete genome sequences despite strong evi- immunogenic proteins which could affect early feeding
dence for ‘off-target-effects’ demonstrated in model organ- events including pathogen transmission, with no further
isms as well as ticks (83, 84). Model genome sequences have testing reported (90). A RV approach for soft ticks has
also been mined to identify homologues in tick species of been described as a concept paper only (91).
interest. For example, the investigation of R. (B.) microplus The most researched tick species in terms of RV
RNAi phenotypes utilized Drosophila melanogaster (fruit approaches has focused on the identification of vaccine
fly) homologues for cellular viability (85). In addition, to candidates to protect against the cattle tick R. microplus
identify proteins associated with the tick RNAi pathway, fly species complex (including Rhipicephalus australis and
and nematode homologues were used to mine R. microplus specific clades of R. microplus and R. annulatus) (92;
EST sequences (86) and available I. scapularis genome Table 2). These ticks collectively vector babesiosis and
reads. Thirty-one putative proteins were identified including anaplasmosis to susceptible cattle across cattle popula-
a putative tick Dicer, RISC-associated proteins and an tions worldwide causing widespread losses (93, 94). One
RNA-dependent RNA polymerase not previously identified approach mined the BmGI EST database available in
in arthropod species (85). 2007 and sequences from subtraction library experiments
(86, 95) for specific Pfam database (96) signatures of
putative antigenic proteins (97). Localization of the Pfam
Predictive algorithms for RV based tick vaccine
identified 400 ESTs included qRT-PCR analysis of differ-
candidate identification
ent tick stages and organs collected from susceptible and
Reverse vaccinology is defined as the screening of all open resistant breeds of cattle and secretion predictions (Sig-
reading frames that a particular pathogen can express at nalP), followed by the prediction of linear B-cell binding
Table 2 Reverse genetics and high-throughput reverse vaccinology approaches applied for tick vaccine candidate identification
Data or vaccine
Species Reverse genetics approaches output References
Ixodes scapularis Linear predictions of B-cell epitopes in salivary proteins (BCPREDS) Not tested (89)
Phage display of 24 h tick saliva expressed genes screened using antibodies 182 contigs identified, (90)
No further testing
Ornithodoros Secreted salivary proteins and concealed mid-gut antigens shown to be critical Review paper (91)
soft ticks to the function of the tick; low copy number gene; and shared conserved
epitopes among several tick species
Rhipicephalus Gene discovery (EST library, subtraction libraries, microarray analysis, Ongoing (97)
(Boophilus) CattleTickBase), Bepipred predictions of linear B-cell epitopes, qRT-PCR
microplus screening, predicted SignalP and localization screening, anti-B-cell peptide
antibody in vitro tick feeding
EST library, DNA microarrays, membrane and secreted proteins, VaxiJen, Ongoing (101)
mouse antiserum screening
Systems biology, CattleTickBase, RNA interference screening, in silico 75% protection (40)
antigenicity

epitopes using Bepipred (97, 98). Antibodies produced

VACCINOMICS APPROACH TO TICK
against peptides recognized by tick resistant breeds of
VACCINES
cattle were fed to semi-engorged female ticks in vitro
(99), and the corresponding peptides of the antibodies The challenge of developing improved vaccines against ticks
causing damage to ticks fed in vitro are currently being and transmitted pathogens arises from the need to under-
tested in cattle vaccine trials (97). Another approach was stand the complex molecular relationship between verte-
based on a ‘systems biology’ analysis of transcriptome brate hosts, ticks and pathogens which requires a systems
data sets and RNAi screening of potential candidates biology approach that will allow the integrated analysis of
identifying an aquaporin protein found to be protective metabolomics, transcriptomics, proteomics and other omics
against R. annulatus and R. microplus challenge at 68% data for discovery of key pathways and molecules that medi-
and 75%, respectively (40, 100). A third approach utilized ate tick–host–pathogen interactions. A vaccinomics
the screening of the same BmGI EST database (86) using approach could then be used to identify and fully character-
a custom microarray to screen different stages and ize candidate protective antigens in different tick and patho-
organs of ticks. Functional annotations were undertaken gen species and validate vaccine formulations.
prior to analysis using VaxiJen, which was developed to
predict protective antigens of bacterial, viral and tumour
Towards an integrated systems biology approach for tick
origin (101). Mouse antisera were used to prescreen the
vaccine development
putative candidates, which are currently being tested in
cattle vaccine trials. Vaccinomics in this article refers to a holistic approach
Limited uses of algorithms have been applied for the based on the use of genome-scale or omics technologies
identification of putative tick vaccine antigens using the and bioinformatics in a systems biology approach for the
above RV approaches. A limitation in the use of linear B- development of next-generation vaccines (107). The scien-
cell predictions above (BCPREDS and Bepipred) is based tific principle underlying a systems biology approach to
on the fact that up to 90% of B-cell epitopes are known tick vaccine development is that ticks, hosts and pathogens
to be conformational and not linear (102). With R. mi- have evolved molecular interactions that affect their sur-
croplus EST resources used (97, 101), many of the vival and life cycle and this co-evolution involves genetic
sequences would have been incomplete; thus, the determi- traits of the tick vectors, vertebrate hosts and pathogens.
nation of conformational B-cell epitopes was not possible Vaccinomics is therefore seen as translational research, in
without access to a fully annotated tick genome. Data- which basic biological information on tick–host–pathogen
bases such as AgAbDb have been established based on interactions translates into the identification and subse-
co-crystal structures to assist to determine conformational quent evaluation of new vaccines. The pipeline and the
B-cell epitope structures (103). Many predictive tools have methods proposed for tick vaccinomics were described
been developed to predict protein domains, secretion, recently (107, 108) (Figure 2).
transmembrane helices, GPI (Glycosylphosphatidylinositol The systems biology approach to the characterization of
(GPI anchor)) anchor, cellular localization and immune the molecular mechanisms that mediate tick–host–patho-
binding epitopes (104, 105). There is a lack of ectoparasite gen interactions identifies host/tick response and infection-
specific tools for immuno-informatics analyses that have transmission factors and will likely provide new targets for
been tested or confirmed, with most tools reported to date the control of tick infestations and/or pathogen infection
developed based on bacterial and viral antigen discovery. and transmission (107). Omics data integration should be
Reverse vaccinology tick research approaches have also followed by the identification of candidate protective anti-
been limited by the absence of fully annotated tick gen- gens using a combination of different screening platforms
omes. In addition, it is predicted that ticks have ~20 000 to formulate and test vaccines in controlled pen trials and
coding genes compared to ~3000 (average) for bacteria. under field conditions (107). Finally, the biological func-
For bacteria, the comparison of multiple genomes (pan- tion and protective mechanisms elicited by vaccination
genomics) has been recently added to RV pipelines to should be characterized to provide additional information
improve the robustness of antigen predictions (106). Per- to design improved vaccines based on the combination of
haps the future for tick vaccines will be the analysis of antigens with different functions to produce a synergistic
several genomes within and between species, to develop effect upon vaccination (107). The integration of omics
novel generic globally applicable tick vaccines. As genome databases for the identification of candidate protective
sequencing technologies continuously improve, there are antigens and the characterization of the immune mecha-
many options to consider for the future of tick vaccine nisms by which protective and nonprotective responses to
development. vaccines are generated are the major challenges in

Figure 2 Vaccinomics pipeline for the development of vaccines for the control of tick infestations and pathogen infection/transmission.
Vaccinomics refers to a holistic approach based on the use of genome-scale or omics technologies and bioinformatics for systems biology
analysis of host–tick–pathogen interactions for the development of next-generation vaccines.
vaccinomics (107, 109). Recent results using the Ixodes as involved in tick–pathogen interactions (107). These
scapularis–host–Anaplasma phagocytophilum model illus- proteins were selected as candidate protective antigens
trated the possibilities of these new technologies for the based on their putative role in tick–Anaplasma marginale
identification of key molecules and pathways involved in (Silk (117)), tick–Babesia bigemina (Trospa (118)) and
tick–host–pathogen interactions. tick–pathogen (Subolesin (119)) interactions. First, tran-
scriptome analyses were combined with RNAi studies to
Characterization of I. scapularis–host– identify Silk and Trospa as candidate protective antigens
A. phagocytophilum interactions (117, 118). Tick Subolesin, the ortholog of insect and ver-
tebrate Akirin, was discovered in 2002 as a tick protective
Transcriptomics and proteomics have been used for the char-
antigen in I. scapularis by expression library immunization
acterization of I. scapularis–host–A. phagocytophilum molec-
in a mouse model of tick infestations (120). Both tran-
ular interactions (18, 110, 111). However, only recently the
scriptomics and RNAi experiments have shown its role as
integration of additional data sets from metabolomics studies
a transcription factor required for the regulation of differ-
has been applied to the study of the I. scapularis–A. phagocy-
ent processes including the innate immune response to
tophilum interface (112–116). The results of these analyses
pathogen infection (119, 121, 122). Then, in vitro capillary
showed that A. phagocytophilum has evolved common
feeding was used to characterize their potential as antigens
molecular mechanisms to establish infection in tick vectors
for the control of both cattle tick infestations and infec-
and vertebrate hosts (111). These strategies include, but are
tion with A. marginale and B. bigemina (123). Recombi-
not limited to, remodelling of the cytoskeleton, inhibition of
nant proteins were produced in Escherichia coli, purified
cell apoptosis, manipulation of the immune response and the
and used to generate antibodies in rabbits. Purified rabbit
use of rickettsial proteins for infection and manipulation of
polyclonal antibodies were added to uninfected or infected
tick and host gene expression (111).
bovine blood to capillary-feed female R. microplus ticks.
Capillary-fed ticks ingested antibodies added to the blood
Vaccinomics approach for the identification of candidate
meal and a reduction in tick weight and/or oviposition
tick protective antigens
was shown when compared with controls fed with blood
For the proof of concept of the vaccinomics approach in alone (123). However, no effect was observed on pathogen
ticks, a pipeline was designed based on proteins identified DNA levels in capillary-fed ticks (123). Nevertheless, these

results together with previous results of functional studies (128–133). In ticks, gene duplications are most likely one
by RNAi suggested that these proteins are promising vac- of the strongest driving forces (125–128, 131, 133). There-
cine antigens for the control of R. microplus infestations fore, our understandings of tick evolution, protein struc-
and infection with A. marginale and B. bigemina and sup- ture and function are vital in the search of new and
ported the conduction of vaccine trials to validate these improved tick vaccines. Furthermore, current data indicate
antigens (9). Vaccination studies showed that vaccination that hard and soft ticks adapted independently and that
with Silk and Subolesin, but not Trospa, reduced tick the large protein families seem to be lineage specific expan-
infestations and oviposition with respect to ticks fed on sions that occurred after their divergence. This strongly
placebo-treated control cattle (124). Furthermore, the suggests that a universal tick vaccine is nonviable and that
results also showed that vaccination with Trospa and Sub- focus should be placed on finding targets within subsets of
olesin reduced B. bigemina DNA levels in ticks while vac- closely related tick genera or even species. Cautions should
cination with Silk and Subolesin resulted in lower thus be placed on extrapolating the protective abilities of a
A. marginale DNA levels when compared with ticks fed specific antigen between tick species, as was seen in the
on placebo control cattle (124). case of Bm86 vaccines (134–139) and a vast number of
These results prove the validity of the vaccinomics antigens that failed to protect against different tick species
approach to select protective antigens and show that vacci- across different hosts (as summarized in Ref. (140)).
nes using tick proteins involved in vector–pathogen inter- Evaluation of all animal vaccine trials carried out to date
actions could be used for the dual control of tick indicates that single antigens display only some 40–60% pro-
infestations and pathogen infection. Remarkably, vaccina- tection under controlled conditions (140). From experience,
tion with Subolesin has shown an effect on the reduction we know that these levels of protection will be greatly
of infestations by different ectoparasites and infection by reduced under field conditions. This should stress the need
different pathogens, therefore suggesting the possibility of to develop combinatorial tick vaccines and to expand our
developing vaccines for the control of multiple ectopara- ideas on what a ‘targetable’ genome may be in ticks. For
sites and infection/transmission of vector-borne pathogens example, a proteome’s complexity arises not only from the
(6). Additionally, vaccinomics approach also results in the proteins three-dimensional structure (lower level informa-
identification of pathogen-derived candidate protective tion), but also from its functional interactions (higher level).
antigens (116). In this way, the combination of tick- This suggests an organized modularity for a proteome with
derived with pathogen-derived antigens might results in connections to regulators, mediators and adaptors, all open-
more effective vaccines for the control of TBDs (6). ing up a huge new complexity for therapeutic intervention.
We are entering an era where our understanding of how
ticks evade vaccination and immune responses with their
INSIGHT FROM TICK PROTEIN EVOLUTION:
highly repetitive genomes, and how to formulate our vacci-
TARGETING OF BINDING SPECIFIC EPITOPES
nes to ensure that protective epitopes remain accessible is
(PPIS) AND CONSERVED METABOLIC
vital. To date, only two examples of promising combinato-
PATHWAYS
rial vaccines are known that of BM86 and subolesin (6,
From a previous section of this article describing the gen- 107) and the peptide vaccine derived from a RV approach
omes sizes and repetitive nature of tick genomes, one is (Table 2) (41). Although not fully understood, the func-
faced with questions pertaining to the origin(s) and signifi- tions of BM86 and Subolesin are most likely to be differ-
cance of these large tick genomes and its impact on vac- ent based on the phenotypes observed after vaccination
cine design. Leading studies from the group of Ben J with individual antigens and the subcellular localization of
Mans have provided significant evidence for not only gene the antigens. Based on this consideration, a new vaccine
duplication leading to protein superfamilies, but also evi- formulation containing a combination of BM86 and Subo-
dence that at least one genome duplication event occurred lesin was recently patented claiming synergy between these
for ticks (125–127). Evidence is growing on how rapid antigens resulting in higher vaccine efficacy against cattle
ticks can adapt to new climates, host species and chal- tick infestations (6). The true mechanism underlying the
lenges set by the use of acaricides, stressing their ability to protective ability of the latter combination, as well as the
acquire novel properties and functions within a short per- RV-derived combination, remains to be elucidated.
iod of time. The adaptability of parasitic organisms has
been ascribed to various factors including gene duplica-
Protein–protein interactions and metabolic pathways
tion, molecular chaperones, antigenic variation, polymor-
phisms, molecular mimicry, rapid turnover of surface It is well known that protein sequences are more con-
molecules and gene disruption to name but a few served than their coding nucleotide sequences, and that

protein folds are more conserved than the amino acid expressed in R. appendiculatus (155). No characterization
sequences encoding them. Even more conserved are the of proteins that cross-react with immune sera (as is the
active site residues of enzymes and the binding specific case with sera raised against subolesin that show signifi-
epitopes occurring in PPIs. Proteins function not merely cant cross-reactivity) has been attempted. If one considers
as single entities, but display their roles by interacting with gene duplications in tick genomes, tick proteins may well
other cellular components, as seen in the case of signalling make use of ‘misleading’ surfaces with immunogenic epi-
pathways and signal transduction, cytoskeleton arrange- topes, which will not necessarily be protective epitopes.
ment and re-arrangement, biological recognition in the This raises questions to the number of antigens that failed
immune system, enzyme regulation and gene expression in animal trials and are being discarded as potential vac-
(141). In ticks, transcriptome analyses have identified pro- cine candidates.
teins acting in almost all of the latter pathways, but no In ticks, only a single yeast two-hybrid study has been
studies have been performed to validate core pathways conducted, resulting in the identification of binding-part-
and to better understand the PPIs occurring in these path- ners of subolesin (121). Since then, additional cDNA
ways. Without this information, our understanding of tick libraries have been cloned into various two-hybrid bait
biology and number of vaccine candidates and drug tar- and prey plasmids, resulting in the identification of addi-
gets that can be evaluated remains inefficient. tional PPIs in R. microplus (patents pending). The protec-
In the case of developing vaccines against parasites such tive ability of these protein–protein interacting antigens is
as Plasmodium (the causative agent of malaria), Mycobac- currently being evaluated in cattle vaccine trials. Recently,
terium tuberculosis and Toxoplasma (the causative agent surface plasmon resonance was used to confirm the PPI
of toxoplasmosis), PPIs have shaped the field significantly between boophilin from R. microplus and thrombin (156).
as it filled major gaps in the knowledge of parasite biology In combination with the available crystal structure data
and provided new vaccine targets, screens for parasite–host (157), this opens an exciting era to evaluate the use of pro-
interactions and defining protective vs. decoy conforma- tein binding epitopes as vaccines and to screen peptide
tional epitopes which may have a tremendous impact on and chemical libraries for small molecules or peptides cap-
the potential of antigens as protective vaccine candidates able of disrupting the PPIs between boophilin and throm-
(Figure 3) (142–151). To date, only limited studies on bin.
Bm86 have evaluated epitopes as being protective or decoy With regard to PPIs involved in vector–pathogen–
epitopes (152–154). Only one study on the mechanism(s) mammalian host interphases, the Borrelia burgdorferi
used by ticks to overcome Bm86 vaccination is available, field (causative agent of Lyme disease) is setting the
indicating that homologous Bm86 coding areas are trend with interactions described for BB0323 and
Figure 3 Schematic presentation of immunogenic epitopes and protein binding epitopes. A tick attached to the host secrets a number of
salivary gland proteins into the feeding pool. Outside of the feeding pool, blood coagulation can occur as normal (cluster of purple and
red cells). The effect of generating antibodies against immunogenic epitopes (yellow boxes) is shown for one of the tick proteins, as well as
the ability of the tick protein to remain biological active bind continuous binding to its host target protein (host factor). The protein–
protein binding sites (PPIs) are indicated with orange boxes. By targeting these PPIs, it may be possible to disrupt biological activity of tick
salivary gland proteins. The same principle can apply to PPIs occurring inside the tick tissues.

BB0323 (158, 159) as well as OspA and CD40 (160).

CONCLUSIONS AND FUTURE DIRECTIONS
Additional targets of interest include the tetratricopep-
tide repeat domain of BB0238 (161) and La7 (162). Cur- Tick vaccines to date focused on targeting important bio-
rently, interactomes of tick tissues of interest for logical processes (predominantly tick attachment and
vaccination as well as the tick–pathogen and tick–host digestion) with the use of single antigens. Although these
interphases remain unexplored. As most tick proteins are studies provided much needed insight into tick biology, it
not annotatable using standard computational tools, the failed to provide full protection against tick infestations.
use of in silico method to predict PPIs will may of lim- Based on our growing knowledge of tick genome complex-
ited use and we may have to rely on classical techniques ity and protein superfamilies, we are entering a new era
such as two-hybrid, pull down assays, antitag-mediated that will require of us to understand the mechanism used
co-immunoprecipitation, peptide arrays, large-scale co- by ticks to overcome changes in their environments (such
immunoprecipitation studies and mass spectrometry to as in the presence of vaccines or acaricides) and to expand
initiate the field. For predicting pathogen–host interac- our knowledge on the ‘targetable’ genome of ticks. In this
tions, in silico methods may prove valuable, as support- regard, RV, PPIs (especially those occurring across tick
ing large data sets are available to be used as scaffolds species) as well as systems biology used in vaccinomics will
(146, 163–166). be vital.
In addition to PPIs, another strategy to improve cur- Studies on ticks and the tick–host–pathogen interactions
rent vaccines involves the simultaneous targeting of anti- suggest that a combination of pathogen-derived ligands
gens that function within a conserved metabolic and/or tick/host receptors and other tick protective anti-
pathway that occur in related tick species. In ticks, this gens will be needed in vaccines (6). In this way, the vaccine
has been evaluated to some extend by targeting gut pro- containing vector and pathogen-derived antigens may pro-
teins involved in tick digestion (as described in this arti- tect vertebrate hosts against tick infestations and pathogen
cle). In 2015, a study by van Zyl et al. attempted to infection while also decreasing pathogen transmission and
identify transcripts involved in metabolic pathways that therefore the risk of disease in humans and animals.
are conserved in both R. microplus and Rhipicephalus
decoloratus gut tissue. Chi-square analysis revealed that
ACKNOWLEDGEMENTS
genes involved in lipid transport and metabolism are sig-
nificantly overrepresented in both species (167). Since We thank members of our laboratories for fruitful discus-
then, these data were subjected to metabolic pathway sions. We acknowledge Martina Hajduskova (www.bi-
analysis and key antigens selected, produced using ographix.cz) for the design of Figure 1 and Dr. Christian
recombinant E. coli and shown to be immunogenic. Vac- Stutzer for the design of Figure 3. The preparation of this
cine trials using these combinatorial antigens are in pro- chapter was partially supported by the European Union
cess. In conclusion, RNA-seq and DNA microarray FP7 ANTIGONE project number 278976. P.K. was sup-
analyses have identified tick proteins involved in a num- ported by the Czech Science Foundation, Grant. No. 13-
ber of metabolic pathways all of which remains to be 11043S. CMO is supported by the National Research
evaluated for PPIs and possible combinatorial vaccines Foundation of South Africa and the Technology Innova-
targeting conserved metabolic pathways. tion Agency of South Africa.
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Distintos Blancos Contra Ticks

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Parasite Immunology, 2016, 0, 1–16 DOI: 10.1111/pim.

Commissioned Review Article

Strategies for new and improved vaccines against ticks and

SUMMARY Keywords interactomics, reverse genetics, systems biology,

© 2016 John Wiley & Sons Ltd 1

Figure 1 An overview of tick physiological

2 © 2016 John Wiley & Sons Ltd, Parasite Immunology, 0, 1–16

© 2016 John Wiley & Sons Ltd, Parasite Immunology, 0, 1–16 3

4 © 2016 John Wiley & Sons Ltd, Parasite Immunology, 0, 1–16

© 2016 John Wiley & Sons Ltd, Parasite Immunology, 0, 1–16 5

Species Life stages sequenced Technology Output References

6 © 2016 John Wiley & Sons Ltd, Parasite Immunology, 0, 1–16

© 2016 John Wiley & Sons Ltd, Parasite Immunology, 0, 1–16 7

epitopes using Bepipred (97, 98). Antibodies produced

8 © 2016 John Wiley & Sons Ltd, Parasite Immunology, 0, 1–16

© 2016 John Wiley & Sons Ltd, Parasite Immunology, 0, 1–16 9

10 © 2016 John Wiley & Sons Ltd, Parasite Immunology, 0, 1–16

© 2016 John Wiley & Sons Ltd, Parasite Immunology, 0, 1–16 11

BB0323 (158, 159) as well as OspA and CD40 (160).

12 © 2016 John Wiley & Sons Ltd, Parasite Immunology, 0, 1–16

© 2016 John Wiley & Sons Ltd, Parasite Immunology, 0, 1–16 13

14 © 2016 John Wiley & Sons Ltd, Parasite Immunology, 0, 1–16

© 2016 John Wiley & Sons Ltd, Parasite Immunology, 0, 1–16 15

16 © 2016 John Wiley & Sons Ltd, Parasite Immunology, 0, 1–16

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